Opening batsmen Joe Burns and Matt Renshaw were dismissed for four and eight respectively while Peter Handscomb was out first ball for nought to leave Australia reeling at 38 for three.
The trio were playing in place of Cameron Bancroft, David Warner, the vice-captain and Steve Smith, the skipper. They were all banned and sent home after for their roles in the ball-tampering plot during the third Test in Cape Town last weekend.
"Putting three guys into a Test match - two have come from Australia and one hasn't played in a game for a while - was always going to be a little bit tough," said Australia bowling coach David Saker.
"We've tried to really care for each other this week. We're realists and we're going to go out and try and play as well as we can and to put in a performance that the Australian public and our group are proud of.
"So far it hasn't happened," Saker added. "But the effort has been there. There's no doubt that the guys are trying their hardest. It just hasn't worked in this game."
Usman Khawaja resisted gamely for just over two hours. The 31-year-year old faced 84 deliveries during his classy 53. He and Shaun Marsh frustrated South Africa's progress with a fourth wicket stand of 52.
But any ambitions of a spirited fightback were extinguished when wicketkeeper Quinton de Kock made an outstanding catch off the bowling of Vernon Philander to dismiss Khawaja. It was Philander’s third wicket of the innings and he finished the day yielding only 17 runs off 12 overs.
Kagiso Rabada, Morne Morkel and Keshav Maharaj claimed the other Australian scalps to leave the tourists on 110 for 6.
South Africa, who made 488 in their first innings, lead the four Test series 2-1 and will be favourites to wrap up another victory and beat Australia in a home series for the first time since 1969-70.