The human zinc metalloprotease ZMPSTE24 is an integral membrane protein critical for the final step in the biogenesis of the nuclear scaffold protein lamin A, encoded by LMNA. After farnesylation and carboxyl methylation of its C-terminal CAAX motif, the lamin A precursor, prelamin A, undergoes proteolytic removal of its modified C-terminal 15 amino acids by ZMPSTE24. Mutations in LMNA or ZMPSTE24 that impede prelamin A cleavage cause the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) and the related progeroid disorders, mandibuloacral dysplasia type B (MAD-B), and restrictive dermopathy (RD). Here we report a "humanized yeast" system to assay ZMPSTE24-dependent cleavage of prelamin A and examine the eight known disease-associated ZMPSTE24 missense mutations. All show diminished prelamin A processing and fall into three classes, with defects in activity (Class I), protein stability (Class II), or both (Class III). Class II ZMPSTE24 mutants can be rescued by deleting the E3 ubiquitin ligase Doa10, involved in ER-associated protein degradation of misfolded membrane proteins, which may have therapeutic implications. We also show that ZMPSTE24-mediated prelamin A cleavage can be uncoupled from the recently discovered role of ZMPSTE24 in the clearance of ER membrane translocon-clogged substrates. Together with the crystal structure of ZMPSTE24, this "humanized yeast system" can guide structure-function studies to uncover the mechanisms of prelamin A cleavage, translocon unclogging, and membrane protein folding and stability.